A Study of the Differential Expression Profiles of Keshan Disease Lncrna/Mrna Genes Based on RNA-Seq

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Original Article

A study of the differential expression profiles of Keshan disease lncRNA/mRNA genes based on RNA-seq

Guangyong Huang1, Jingwen Liu2, Yuehai Wang1, Youzhang Xiang3

1Department of Cardiology, Liaocheng People’s Hospital of Shandong University, Liaocheng, China; 2School of Nursing, Liaocheng Vocational & Technical College, Liaocheng, China; 3Shandong Institute for Endemic Disease Control, Jinan, China Contributions: (I) Conception and design: G Huang, Y Xiang; (II) Administrative support: G Huang, Y Wang; (III) Provision of study materials or patients: G Huang, J Liu, Y Xiang; (IV) Collection and assembly of data: G Huang, J Liu; (V) Data analysis and interpretation: J Liu, Y Wang; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Guangyong Huang, MD, PhD. Department of Cardiology, Liaocheng People’s Hospital of Shandong University, No. 67 of Dongchang Street, Liaocheng 252000, China. Email: [email protected].

Background: This study aims to analyze the differential expression profiles of lncRNA in Keshan disease (KSD) and to explore the molecular mechanism of the disease occurrence and development. Methods: RNA-seq technology was used to construct the lncRNA/mRNA expression library of a KSD group (n=10) and a control group (n=10), and then Cuffdiff software was used to obtain the gene lncRNA/ mRNA FPKM value as the expression profile of lncRNA/mRNA. The fold changes between the two sets of samples were calculated to obtain differential lncRNA/mRNA expression profiles, and a bioinformatics analysis of differentially expressed genes was performed. Results: A total of 89,905 lncRNAs and 20,315 mRNAs were detected. Statistical analysis revealed that 921 lncRNAs had obvious differential expression, among which 36 were up-regulated and 885 were down- regulated; 2,771 mRNAs presented with obvious differential expression, among which 253 were up-regulated and 2,518 were down-regulated, and cluster analysis indicated that the gene expression trends among the sample groups were consistent. The differentially expressed lncRNAs were tested for target genes, and 117 genes were found to be regulated by differential lncRNAs, which were concentrated in six signaling pathways, among which the apoptosis FoxO signaling pathway ranked first, so the target genes IGF1R and TGFB2 were screened out. Conclusions: In this study, RNA-seq technology was used to obtain the differential gene expression profiles of KSD, and bioinformatics analysis was performed to screen out target genes, pointing out the direction for further research into the etiology, pathogenesis and drug treatment targets of KSD.

Keywords: Keshan disease (KSD); long noncoding RNA; messenger RNA; RNA sequencing

Submitted Aug 27, 2020. Accepted for publication Nov 29, 2020. doi: 10.21037/cdt-20-746 View this article at: http://dx.doi.org/10.21037/cdt-20-746

Introduction manifestations are ventricular enlargement and a decrease in myocardial contractility, which often causes heart failure, Keshan disease (KSD) is an endemic cardiomyopathy unique arrhythmia and even sudden death. Thus, the prognosis is poor. to China, and patients are mainly found in the narrow and The cause of KSD is not yet fully understood, but low-selenium zone from the northeast to the southwest of research results confirm there exists a close association China. According to statistics (1), 16 provinces (regions), between low selenium levels and KSD (2,3). As an essential with a population of 60 million, are affected. KSD is mainly trace element, selenium realizes its biological function characterized by myocardial involvement, and the clinical mainly through selenoproteins. At present, a large number

Table 1 Clinical data of the KSD group and control group NYHA Number Gender Systolic blood Diastolic blood Heart rate Group Age (Y) LA (mm) LV (mm) LVEF (%) classification of cases (M/F) pressure (mmHg) pressure (mmHg) (times/min) (I/II/III/IV)

KSD group 10 4/6 50.9±7.3 116.6±16.0 74.6±10.0 73.9±11.3 40.6±3.0* 59.7±2.9* 35.6±4.6* 0/7/3/0*

Control group 10 5/5 45.5±4.9 121.0±5.2 75.8±4.2 68.1±4.4 32.0±1.9 45.6±2.2 67.8±3.2 10/0/0/0

t or χ2 0.20 1.94 0.83 0.35 1.51 7.66 12.25 18.17 40.0 Compared with control group, *, P<0.01. LA, left atrial diameter; LV, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; NYHA, American College of Cardiology; KSD, Keshan disease.

of studies have revealed that selenium deficiency is closely analyzed by means of bioinformatics, providing a theoretical related to the occurrence and development of KSD. The basis for shedding light on the mechanism of disease at the selenium content of the environment of the KSD area, level of gene expression regulation. as found in the soil, local food, and hair and blood of the We present the following article in accordance with people, and the plasma selenoprotein P are lower than those the MDAR reporting checklist (available at: http://dx.doi. in non-affected areas (4,5). A systematic review and meta- org/10.21037/cdt-20-746). analysis of selenium deficiency and KSD revealed that the supplementation of selenium could significantly reduce the Methods incidence of KSD (2). Current studies suggest that the mechanism of chronic Clinical data KSD is mainly related to mitochondrial oxidative damage, Ten KSD patients in the severe KSD area of Shandong apoptosis and fibrosis regulation (6-8). Low selenium can Province were enrolled as the KSD group. There were five cause damage to myocardial mitochondria, and selenium male patients and five female patients, ranging from 38 to deficiency can lead to a decrease in the synthesis of 45 years of age, with an average age of 42.6 years. All the glutathione peroxidase. This then leads to the aggravation patients had their medical histories taken, and underwent of oxidative stress damage, in turn causing myocardial a physical examination, electrocardiography, X-ray damage and, ultimately, resulting in cardiomyopathy. photography, cardiac ultrasound and related biochemical Selenium deficiency may also amplify other pathogenic examinations, and the examination results met the factors, including viral and other infections (3). Recent diagnostic criteria of KSD (GB17021-1997). In addition, comparative studies on the gene expression of KSD 10 healthy people, five males and five females, were and dilated cardiomyopathy have found that KSD and enrolled locally as the control group, having undergone a dilated cardiomyopathy have a different pathogenesis: the comprehensive physical examination to confirm that they pathogenesis of KSD mainly manifests as viral infection, had no heart disease. The clinical data of the KSD group cell membrane damage and oxidative stress (9). and control group are presented in Table 1. The present In recent years, the advances in RNA sequencing study was conducted in accordance with the Declaration of technology mean that it has developed into a powerful Helsinki (as revised in 2013) and was approved by the ethics research platform indispensable for exploring diseases. committee of Liaocheng People’s Hospital (No. 201510), At present, it is widely used for research into a variety of and all patients signed written informed consent. complex human diseases, such as malignant tumors, immune diseases and unknown infectious diseases, and it is crucial Main reagents and instruments to the search for pathogens and biomarkers, the exploration of pathogenesis, and the formulation of targeted treatment Main reagents: Trizol kit (Invitrogen life technologies, programs (10). In this study, RNA-sequence (RNA-seq, a USA), DEPC-treated water (Invitrogen life technologies, transcriptome sequencing technology) was used to construct USA), RNase inhibitor (Epicentre, USA), SuperScriptTM differential expression profiles of lncRNA in the peripheral III reverse transcriptase (Invitrogen, USA), 5× RT buffer blood plasma of KSD patients. These profiles were then (Invitrogen, USA), 2.5 mM dNTP mixture (HyTest

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Ltd., Finland). Main instruments: NanoDrop ND-2000 to remove rRNAs from the RNA, and a TruSeq Stranded instrument (Thermo Fisher Scientific, USA), Ribo-Zero Total RNA Library Prep Kit (Illumina, USA) was used to rRNA Removal kits (Illumina, USA), TruSeq Stranded pre-process the RNA to construct a sequencing library. A Total RNA Library Prep Kit (Illumina, USA), BioAnalyzer BioAnalyzer 2100 instrument (Agilent Technologies, USA) 2100 instrument (Agilent Technologies, USA), Illumina was used for library quality control and quantification. In HiSeq4000 (Illumina, USA). accordance with the Illumina sequencing instructions, the 10 pM library was denatured into single-stranded DNA molecules, which were captured on an Illumina flow cell Experimental methods and amplified in situ into clusters. They then underwent Specimen acquisition 150 cycles of sequencing on the Illumina HiSeq sequencer. The subjects fasted for 12 hours, and blood was withdrawn from the elbow vein in the morning, after they had got up. Statistical analysis Five mL of peripheral venous blood was withdrawn from each subject in both the KSD group and the control group, Original data was obtained after sequencing by an Illumina with an anticoagulation negative pressure tube, and stored HiSeq4000 sequencer. The first quality control of the at −80 . raw data was performed using the Q30 value. Cutadapt software (v1.9.3) was used to remove adapters and low- ℃ RNA extraction quality reads, and then Hisat2 software (v2.0.4) was used The blood sample was removed from the −80 under the guidance of an Ensembl gtf gene annotation refrigerator, thawed, and centrifuged at 12,000 g at 4 for file to align high-quality adapter-free reads to the human ℃ 10 minutes to remove impurities. Next, 250 μL of the blood reference genome (UCSC MM10). Cuffdiff software (v2.2.1) ℃ was taken and transferred to a 1.5-mL centrifuge tube, was used to obtain a gene-level lncRNA/mRNA FPKM 750 μL of TRIzol LS reagent and 20 μL of glacial acetic (Fragments Per Kilobase of transcript per Million fragments acid were added, and the tube was shaken to mix the mapped) value, as the lncRNA/mRNA expression profile. solution up well. After homogenization, the sample was Agilent Gene Spring software was used for data analysis, incubated at 15 to 30 for five minutes, 0.2 mL of and standardized processing was performed to obtain two chloroform was added, the tube was vigorously shaken for ℃ sets of fold change values. After statistical analysis using 15 seconds, incubated at 30 for two minutes, and then a t-test, differential lncRNA/mRNA expression profiles centrifuged at 12,000 g and 4 for 15 minutes. After ℃ were obtained. The standard was that the fold change in centrifugation, the blended liquid had separated out into ℃ the two groups was more than double and P<0.05. Gene layers, with the RNA in the colorless water layer. The upper Spring Software 11.0 was used to do a cluster analysis of the water layer was transferred to another centrifuge tube, differentially expressed lncRNA/mRNA. As a regulatory and 0.5 mL of isopropanol was added to it. The mixture factor, lncRNA can regulate adjacent protein-coding genes, was left to stand at room temperature for 10 minutes, then and target gene detection finds differentially expressed centrifuged at 12,000 g and 4 . After 10 minutes, RNA genes and marks them as target genes. Differential gel-like precipitates could be seen on the bottom and sides ℃ genes were subjected to Gene Ontology (GO) biological of the tube. The upper liquid layer was removed, and function analysis and Kyoto Encyclopedia of Genes and 1 mL of 75% ethanol was added to clean off the gelatinous Genomes (KEGG) pathway analysis. P<0.05 was considered sediment attached to the tube sides. It was fully oscillated, statistically significant. centrifuged at 75,000 g and 4 for five minutes. The upper liquid layer was removed again, and the sediments were left ℃ standing but not to completely dry out. About 5–10 minutes Results later, RNase-free water was added to dissolve the RNA, and Clinical data of the KSD group and the control group the solution was incubated at 55–60 for 10 minutes and, finally, stored at −80 . The differences in gender composition, age, systolic ℃ blood pressure (SBP), diastolic blood pressure (DBP) and ℃ CDNA library preparation and sequencing heart rate between the two groups were not statistically A Ribo-Zero rRNA Removal Kit (Illumina, USA) was used significant; compared with the control group, in the KSD

2,771 KSD 921 CTRL KSD CTRL mRNAs IncRNAs

Differentially expressed IncRNAs Differentially expressed mRNAs

Figure 1 Differential expression profiles of lncRNA/mRNA. KSD, Keshan disease; CTRL, control.

group, the left ventricular end-diastolic diameter and left consistent (Figure 2). The differentially expressed lncRNAs atrial diameter were significantly larger, the left ventricular were tested for target genes, and 117 genes were found to ejection fraction was significantly lower (P<0.01), and the be regulated by differential lncRNA. heart function was significantly lower (P<0.01,Table 1).

Bioinformatic analysis Quality control of RNA and library In order to determine the biological process (BP) and The RNA concentration of each sample was measured by molecular mechanism of differentially expressed genes in NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, KSD, an enrichment analysis of the function and pathway MA, USA), and the OD260/OD280 value was used as an of differentially expressed genes was also carried out. An indicator of RNA purity. In this study, the OD260/OD280 enrichment analysis of the function and pathway was carried range was 1.8–2.1, which is to say, the purity of the RNA out using the database for annotation, the visualization was qualified. RNA integrity was tested using denatured and integrated discovery (DAVID) online tool (11). The agarose gel electrophoresis. It was observed that the tool is based on GO and the KEGG database. GO analysis RNA5s, 18S and 28S bands were clear and intact without is a biological gene bank used to annotate genes, gene any obvious degradation. products and sequences. It is divided into three parts, and determines the molecular function (MF), BP and cell component (CC). In this study, a CO function analysis Differential expression of lncRNA/mRNA of the differential expression of lncRNA was conducted By using the RNA-seq technique, a total of 89,905 lncRNAs to learn more about the function of these lncRNAs, and 20,315 mRNAs were detected (Figure 1). Data analysis GO terms with a P value of <0.05 were considered to be revealed that the difference in the expression of lncRNA statistically significant. GO function analysis revealed that in plasma between the KSD group and the control group the differentially expressed lncRNAs were mainly involved was statistically significant. Statistical analysis revealed that in the BPs of neuronal differentiation, cell morphogenesis 921 lncRNAs had obvious differential expression (fold and differentiation, actin filament binding, superoxide anion change >2, P<0.05), of which 36 were up-regulated and generation and hydrogen peroxide biosynthesis (Figure 3). 885 were down-regulated; 2,771 mRNAs presented with A KEGG pathway analysis was done to map molecular obvious differential expression, of which 253 were up- data sets from genomics, transcriptomics, proteomics and regulated and 2,518 were down-regulated. A cluster analysis metabonomics to a KEGG pathway map for biological of the differential expression of lncRNA was conducted function interpretation, with P≤0.05 as the threshold of and the difference in lncRNA expression between the significant enrichment. The KEGG pathway analysis KSD group and the control group could clearly be seen. showed that differentially expressed lncRNAs were enriched The same group of samples was gathered together, and in six signaling pathways (Table 2), among which the FOXO the results indicated that the gene expression trend was signaling pathway ranked first Figure( 4).

A B

Figure 2 Cluster analysis of the differential expression of lncRNA. (A) Cluster analysis of differentially expressed lncRNA between the KSD group (KSD 1-10) and control group (ctrl1-10), the red part represents the up-regulation of lncRNA, and the green part represents the down- regulation of lncRNA. (B) Cluster analysis of mRNA differentially expressed between the KSD group (KSD 1-10) and control group (ctrl1-10), the red part represents the up-regulation of mRNA, and the green part represents the down-regulation of mRNA. KSD, Keshan disease.

QRT-PCR validation Discussion

The names and the fold changes of the top 20 lncRNA/ KSD was first found in Keshan County, Heilongjiang mRNA are in Table 3. Three significantly up-regulated Province, China, in 1935. After the Second World War, lncRNAs and three significantly down-regulated similar cases were also found in the Nagano Prefecture lncRNAs were randomly selected (ENST00000421185, of Japan and the northern mountainous area of Korea ENST00000410140, TCONS_l2_00004571; down- (12,13). After more than half a century of prevention and regulated: ENST00000355500, ENST00000359888, control, the type of KSD also changed from acute KSD and ENST00000376025). A quantitative real-time polymerase subacute KSD to chronic KSD. In patients with chronic chain reaction (qRT-PCR) was performed on 10 patients KSD, the heart is significantly enlarged and the cardiac with KSD and 10 healthy controls. It was revealed that, function is decompensated. This is mainly characterized by in the expanded samples, the PCR results were basically severe cardiomyopathy and multifocal myocardial necrosis, consistent with the RNA-seq results (Figure 5). lncRNA/ which are the focus of prevention and treatment research at mRNA network is in Figure 6. present. Therefore, in this study, patients with chronic KSD

A Sig GO terms of DE gene-BP B Sig GO terms of DE gene-CC Neuron differentiation Plasma membrane

Generation of neurons Neuron projection

Neurogenesis Cell periphery

Cell morphogenesis involved in differentiation Neuron part

Cell projection organization Cell junction

Cell morphogenesis involved in neuron Cell projection

Neuron development Cell-cell junction

Cellular component disassembly Stress fiber

Neurofilament cytoskeleton organization Actin filament bundle

Neuron projection development Actomyosin

0 1 2 3 4 5 6 0 1 2 3 4 5 6 Enrichment score [−log10(Pvalue)] Enrichment score [−log10(Pvalue)] C Sig GO terms of DE gene-MF Protein binding

Snare binding

Cytoskeletal protein binding

Actin filament binding

PDZ domain binding

Enzyme binding

Insulin receptor substrate binding

Rac guanyl-nucleotide exchange factor activity

tRNA methyltransferase activity

G-protein alpha-subunit binding

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Enrichment score [−log10(Pvalue)]

Figure 3 Functional distribution of differentially expressed lncRNA by GO analysis. (A) GO enrichment analysis of differentially expressed lncRNAs in biological process (BP). (B) GO enrichment analysis of differentially expressed lncRNAs in cell components (CC). (C) GO enrichment analysis of differentially expressed lncRNAs in molecular functions (MF). GO, Gene Ontology.

Table 2 Signaling pathways with enriched differential genes Pathway ID Definition Genes

hsa04068 FoxO signaling pathway—Homo sapiens (human) IGF1R/TGFB2

hsa04390 Hippo signaling pathway—Homo sapiens (human) NKD1/TGFB2

hsa04020 Calcium signaling pathway—Homo sapiens (human) AVPR1B/GRIN2C

hsa05014 Amyotrophic lateral sclerosis (ALS)—Homo sapiens (human) GRIN2C/NEFM/SOD1

hsa04213 Longevity regulating pathway—multiple species—Homo sapiens (human) IGF1R/SOD1

hsa04520 Adherens junction—Homo sapiens (human) IGF1R/PVRL1

Figure 4 FoxO signaling pathway (hsa04068). were chosen as the study subjects, and RNA-seq technology repeatability, a wider detection range and more reliable was used to construct the differential expression profiles of analysis. It uses high-throughput sequencing technology to lncRNA/mRNA in KSD, in order to explore the molecular sequence all cDNA library from RNA reverse transcription mechanism and target genes of KSD at the gene level. in tissues or cells in order to calculate the expression levels At present, research into KSD mainly focuses on of different RNAs by counting the number of related reads. the association between selenium deficiency and KSD. Thus, more comprehensive genetic information, such as However, studies on the mechanism have mainly focused on transcriptional location and splicing, can be determined (10). selenium-related mitochondrial oxidative damage, apoptosis In the present study, a total of 117 genes were found and myocardial fibrosis. Selenium deficiency may also to be regulated by differential lncRNAs. GO function increase the infection of enterovirus and other pathogens, analysis of the differentially expressed genes revealed that through the above-mentioned mechanism, leading to the the differentially expressed lncRNAs were mainly involved occurrence and progress of KSD, and finally causing heart in the BPs of neuronal differentiation, cell morphogenesis failure (3,6-8). In a previous study by this research team, and differentiation, actin filament binding, superoxide miRNA sequencing was also conducted in patients with anion generation and hydrogen peroxide biosynthesis. The dilated cardiomyopathy, and bioinformatic analysis revealed results showed that KSD patients differ from normal people that a neuronal differentiation process and a mitogen- in cardiomyocyte differentiation and oxidative stress. Pei activated protein kinase (MAPK) signaling pathway were et al. (7) found that the level of oxidative stress increased involved in the occurrence and development of dilated significantly and was positively correlated with the degree cardiomyopathy (14). Therefore, there are some differences of myocardial damage in patients with KD. The present in miRNA between KSD and dilated cardiomyopathy. study confirmed the argument that oxidative stress is related RNA-seq can be used to study gene expression to myocardial injury in KSD at the gene expression level. differences at the whole genome level, which has the KEGG pathway analysis demonstrated that the advantages of more accurate quantification, higher differentially expressed lncRNAs were enriched in six

Table 3 The names and the fold changes of the top 20 lncRNA/mRNA EXP vs. CTRL differentially expressed lncRNAs top 20 EXP vs. CTRL differentially expressed mRNAs top 20

Transcript_id EXP_FPKM CTRL_FPKM P value Transcript_id EXP_FPKM CTRL_FPKM P value

ENST00000428088 3.86662 103.259 0.00005 MAP2 1.0499 105.324 0.00005

ENST00000433084 2.35982 49.2815 0.00005 SLC1A3 1.6004 71.4991 0.00005

ENST00000447372 0.313693 18.8243 0.00005 EPB41L2 6.47075 528.989 0.00005

ENST00000461291 0.28308 15.2866 0.0001 MXRA5 0.192223 6.49242 0.0001

ENST00000563424 0.418345 24.5947 0.00015 NDRG4 1.17598 166.429 0.0001

ENST00000565936 0.234962 11.9778 0.00015 CDR2L 0.639103 24.6539 0.00025

ENST00000566968 0.448172 13.5965 0.00015 SLC1A2 0.62356 186.516 0.00025

ENST00000567533 0.489348 23.7616 0.00015 HPCAL4 0.363686 23.0287 0.00035

ENST00000568063 0.263486 16.1909 0.00025 MUC5B 0.517991 16.0292 0.00035

ENST00000566733 0.25072 9.65832 0.00025 PLP1 1.23197 43.0059 0.00035

ENST00000568177 0.432795 19.1586 0.00025 C1orf61 1.6347 101.234 0.00045

ENST00000607352 0.476672 18.412 0.00025 KCNC1 0.630413 97.3282 0.00045

ENST00000608477 0.420748 8.64465 0.0003 UNC13A 0.284624 8.44909 0.0006

TCONS_00001652 46.5416 670.333 0.0003 GFAP 2.33385 205.76 0.00065

TCONS_00009934 0.508349 14.2331 0.00035 PTPN22 9.65913 502.588 0.00065

TCONS_00012197 34.475 977.85 0.00035 KIAA1644 0.288939 15.14 0.00085

ENST00000363624 1121.15 5.54397 0.0004 NAIP 6.29335 0.278693 0.00165

ENST00000363618 201.521 2.64544 0.0005 GIMAP7 17.7951 1.92021 0.00185

TCONS_l2_00004571 71.1207 3.23242 0.0005 MT-ND4L 197.199 5.47898E-18 0.00215

ENST00000459255 149.787 3.07453 0.0005 FOS 206.872 21.6916 0.00265

14 ENST00000421185 12 ENST00000410140 10 TCONS_I2_00004571 ENST00000355500 8 ENST00000359888 6 ENST00000376025

4 Relative expression levels of IncRNA Relative expression 2

0 KSD CTRL

Figure 5 lncRNA (up-regulation: ENST00000421185, ENST00000410140, TCONS_l2_00004571; down-regulation: ENST00000355500, ENST00000359888, ENST00000376025) the results in the expanded samples verified by RT-PCR (*, P<0.05; **, P<0.01; ***, P<0.001). KSD, Keshan disease; CTRL, control.

SLC1A2 C1orf115

ENST00000607352

PFN2 FRMD4A

SOCS5 RPP25 GTF3C1 RAI1 PSD PLXNA2

GAD1 KLK6

ENST00000567533 RIC8B SLC10A2 TP53BP2 SLC8A2 GPR55 BTBD19 C21orf90 CSRP2BP KCTD21 POTEC LINGO1 OR2M2 CDK18 HAND1 ZNF384 PPP2R3A COL12A1 CSPG5 LY6H RHEB NRP1 HCRTR1 DKFZP434O1614 STARD10 GPSM1 GANC HMGA1 SOX11 APTX IRF3 FFAR1 RPTN SYT13 ZC3H18 DLG2 HIST1H1B C13orf35 TRPV6 UBFD1 TTLL11 DIRAS1 KCNJ16 ZFAT OLFM1 SENP8 AKR1C1 SCUBE3 RNF122 IGFBP3 TNF PTGER3 SPINT1 SEC14L6 CTSH SLC6A15 ATP8A1 PSD4 NAT9 ESPNL FAM98C PLEKHA8 STYX USP49 AKAP1 POLG2 MMP14 IFI16 SPATA6L DLG4 ADSSL1 MMP2 AHCTF1 FAM169B TBC1D3G ASTN2 DNAJB5 AGXT MID1 PCDHB3 SLC17A7 TAF1C OR2T3 AC004381.6 ST8SIA1 CAMTA2 SH3PXD2A RP11-770J1.4 AQP6 AURKAIP1 APEX1 DSCAM ZSCAN22 PEX11A SCRT1 SLC5A1 EPS8L1 OSGEP PCDH15 KLHL7 CACNB3 NIPAL4 ELP5 PLEKHH2 FBRSL1 SNRK HOXA3 MAGEB6 RNF157 OSMR SULT2A1 MTMR11 RAPGEFL1 ST18 OR4K14 DCLK2 COL6A2 PDIA5 DPP10 ARHGAP39 IRS1 USP32 CHRNA2 TBCB AGRN LRP2 CELF5 UNC5D GPHN C14orf79 SLC39A14 LNX2 MGST2 APITD1 SRL MTFR1 BEND7 TMEM9B TJAP1 C2orf78 PHC1 CDH1 ACOT11 FRMD1 TAF5 RIMS4 SCAMP4 NOD2 DRP2 SMTN LTBP4 GNG13 D2HGDH CYP26B1 ZNF777 C21orf58 PGAP2 RIMBP2 SPATA25 PRDM16 GJA4 OR4K13 RBM4 TMEM130 ADCY1 RSPH1 TCTN2 B3GNT1 FASTKD5 KLHL26 SPEF2 BABAM1 IKZF2 CABLES1 AADACL4 DENND3 PURG MFSD6L AC138655.1 WDR66 PPAPDC3 ENST00000568177ENST00000566733 GALNTL6 PRDM2 GRM7 LEPREL4 AP3D1 LILRB5 SHMT2 TMEM169 KCNQ5 EFNB1 KLHL15 MOSPD3 RPS8 ENST00000428088 ARHGEF16 HAPLN4 TCONS_00009934 EIF5AL1 SPATA31D1 HOXD8 IMPACT NOVA1 AC092850.1 MT2A TCOF1 FAM189A1 C17orf72 CATSPER2 TENC1 C20orf202 OR2B2 ENST00000433084 WDR91 PCDH12 FLJ00104 PDE7A NEBL NLGN3 EHBP1 C1QL2 C11orf48 GLTSCR2 C1QL3 BBS5 USP13 CTNNA1 PVRL1 GPNMB FAM198A MYO7A POTEG COL6A1 EIF5A DPYSL2 CD33 KDM1B POMC CTU1 PSMA7 MRPL28 GTF3C4 CILP2 BDKRB1 OR11H6 FREM3 JPH4 APCDD1L TMEM151A CDR2L SCAMP5 GPRIN2 PAH UTP15 GRM6 DGCR2 GADL1 OR10G2 TMEM110 AIM1L RABL2B ELAC2 SLC41A1 NPNT SLC9B2 ACTL7B GLCE AMIGO1 TBC1D26 TTLL4 AC006372.1 RD3 SIM2 PPFIA3 ALDH3B2 PPME1 CDKL2 APCDD1 LMO3 OR4C5 IFT88 ZNF667 SLC6A1 PRUNE2 FUT2 CAP2 GPR64 TMPRSS13 XPR1 SRGAP2 ELOF1 SOST LDLR SLIT2 SLFN11 OR7D4 NPIPB4 AC135983.2 TNFRSF10D RP11-429E11.3 MOK TH ZNF648 AP000974.1 DHRS2 DIXDC1 ARFGEF1 SLC39A11 C15orf62 PARVA MUL1 ANKRD6 UGT8 SPSB4 ERN1 RAB44 GAREML SLC27A1 GTF2IRD2B GRIN2C ZBTB40 SPRED2 MSANTD1 PPP1R16A ADAM15 POU3F1 SH3RF2 GPATCH1 GPR174 NCBP2 CLIP3 ARMS2 TMEM98 VRTN TRIM71 SIRT3 ABCA2 GGT1 MZT2B C17orf102 PALM SULF1 MAN1A2 CBX2 KIF17 ABCD2 MED15 TSPAN5 FRZB PATZ1 TERT MIPOL1 CBLN2 EIF3K DRD1 OR2J2 ASCL2 HACE1 GJA1 ABHD4 DNAL1 TMCO3 GRTP1 MEX3A RABGAP1 HOXB9 ARHGEF12 HNRNPCL1 FAM167A FGF23 RP11-1220K2.2 C1orf43 PMP22 NAALADL1 CLYBL ENST00000563424 SMO SMAD9 HSPG2 SOGA3 GIPR SRI COIL CDK16 OR4C13 N4BP3 ATP7B PARD3B C6orf222 TIGD2 SLC25A11

TBC1D13 CNDP1 TM7SF2 NPAP1 MT3ENST00000608477 ITPR1 RAB40C PLXNB1 ENST00000566968 ENST00000461291 TCHH ORC6 ENST00000565936 FAM175AENST00000447372 LRIT2 MED1 USHBP1 IFT46 CCDC71 OR6J1 CLN6 HOOK2 Figure 6 ncRNA/mRNA network for lncRNA-mRNA with Pearson correlation coefficient >0.99. signaling pathways, in which the FoxO signaling pathway, genes detected in the preliminary results were IGF1R and which is related to apoptosis, ranked first. FOXO plays TGFB2. IGF1R participates in the process of myocardial an important role in cell metabolism, cell survival and hypertrophy by changing cell cycle, apoptosis and resistance to oxidative stress (15). It has been confirmed interaction with the surrounding environment. A previous in one study that by increasing the resistance of mice study revealed that the IGF1 level in the myocardium of to oxidative stress, FOXO could reduce myocardial patients with idiopathic hypertrophic cardiomyopathy injury. FOXOs play a critical role in promoting was significantly higher than that in patients with aortic cardiomyocyte survival during conditions of oxidative stress stenosis. Immunohistochemistry and in situ hybridization through the induction of antioxidants and cell survival revealed that IGF1 expression was up-regulated in patients pathways (16). Another experiment verified that FOXO with idiopathic hypertrophic cardiomyopathy. Therefore, expression was down-regulated in mice with oxidative it is suggested that the up-regulation of IGF1 expression is stress-induced myocardial injury (17). This study revealed not the result of myocardial hypertrophy but the cause of that the differentially expressed lncRNAs were significantly myocardial hypertrophy (18). One study induced IGF1R enriched in the FoxO signaling pathway and noticeably silencing with RNA interference, and the results were that down-regulated. It can be inferred that the occurrence the severity of myocardial hypertrophy was significantly and development of KSD may be related to the weakening reduced, and the myocardial function was improved. of myocardial resistance to oxidative stress caused by the TGFB2, another gene screened candidate, is a transforming down-regulation of the FoxO signaling pathway. The growth factor. A further study revealed that, after RXRA intervention of this signaling pathway may provide new gene knockout, TGFB2 gene expression and cardiomyocyte ideas for the treatment of KSD. The key genes affecting the apoptosis increased, and the embryonic cardiac outflow tract occurrence and development of KSD were further screened development was abnormal (19). The investigators believed using the RNA-seq technique, and the two candidate that the abnormal expression of a target gene is related to

© Cardiovascular Diagnosis and Therapy. All rights reserved. Cardiovasc Diagn Ther 2021;11(2):411-421 | http://dx.doi.org/10.21037/cdt-20-746 420 name the occurrence and development of KSD. The next step Attribution-NonCommercial-NoDerivs 4.0 International is to construct a mouse model, with oxidative stress injury, License (CC BY-NC-ND 4.0), which permits the non- selenium deficiency and vitamin deficiency, to verify this commercial replication and distribution of the article with conclusion, using RNA interference induced gene silencing. the strict proviso that no changes or edits are made and the In summary, in this study, RNA-seq technology was used original work is properly cited (including links to both the to construct the differential expression profiles of lncRNA/ formal publication through the relevant DOI and the license). mRNA in KSD, a bioinformatics analysis of differentially See: https://creativecommons.org/licenses/by-nc-nd/4.0/. expressed genes was performed, and IGF1R and TGFB2 were preliminarily screened out as candidate genes. The References investigators will next conduct animal experiments to verify these candidate genes, and continue to study the differential 1. Yan C, Fang W. Status and etiology progress of gene expression and regulation mechanism of KSD by Keshan disease. Advances in Cardiovascular Diseases increasing the sample size, in order to reveal the mechanism 2017;38:225-9. of the occurrence and development of cardiomyopathy of 2. Zhou H, Wang T, Li Q, et al. Prevention of Keshan KSD and, ultimately, facilitate drug treatment. Disease by Selenium Supplementation: a Systematic Review and Meta-analysis. Biol Trace Elem Res 2018;186:98-105. Acknowledgments 3. Loscalzo J. Keshan disease, selenium deficiency, and the We thanked all people who make contributions to this selenoproteome. N Engl J Med 2014;370:1756-60. work. 4. Zhang X, Wang T, Li S, et al. A Spatial Ecology Study of Funding: National Natural Science Foundation of China Keshan Disease and Hair Selenium. Biol Trace Elem Res (81573095). 2019;189:370-8. 5. Zhang X, Wang T, Li S, et al. A spatial ecological study of selenoprotein P and Keshan disease. J Trace Elem Med Footnote Biol 2019;51:150-8. Reporting Checklist: The authors have completed the MDAR 6. Jiang S, Hou J, Liu H, et al. The roles of PPARs reporting checklist. Available at: http://dx.doi.org/10.21037/ and PPARγ coactivator-1 alpha in mitochondrial cdt-20-746 cardiomyopathy. Chin J Endemiol 2018;37:943-6. 7. Pei J, Fu W, Yang L, et al. Oxidative stress is involved in Data Sharing Statement: Available at: http://dx.doi. the pathogenesis of Keshan disease (an endemic dilated org/10.21037/cdt-20-746 cardiomyopathy) in China. Oxid Med Cell Longev 2013;2013:474203. Conflicts of Interest: All authors have completed the ICMJE 8. Ma ZG, Yuan YP, Wu HM, et al. Cardiac fibrosis: uniform disclosure form (available at: http://dx.doi. new insights into the pathogenesis. Int J Biol Sci org/10.21037/cdt-20-746). The authors have no conflicts of 2018;14:1645-57. interest to declare. 9. Huang GY, Xiang YZ, Liu JW, et al. Comparative analysis of lncRNA-mRNA co-expression between Keshan Ethical Statement: The authors are accountable for all disease and dilated cardiomyopathy. Chin J Endemiol aspects of the work in ensuring that questions related 2019;38:361-7. to the accuracy or integrity of any part of the work are 10. Hawkins RD, Hon GC, Ren B. Next-generation genomics: appropriately investigated and resolved. The present study an integrative approach. Nat Rev Genet 2010;11:476-86. was conducted in accordance with the Declaration of 11. Huang da W, Sherman BT, Lempicki RA. Systematic Helsinki (as revised in 2013) and was approved by the ethics and integrative analysis of large gene lists using DAVID committee of Liaocheng People’s Hospital (No. 201510), bioinformatics resources. Nat Protoc 2009;4:44-57. and all patients signed written informed consent. 12. Tan J, Huang Y. Selenium in geo-ecosystem and its relation to endemic diseases in China. Water Air Soil Open Access Statement: This is an Open Access article Pollut 1991;57-58:59-68. distributed in accordance with the Creative Commons 13. Kakehashi Y. Why did Keshan disease occur? The

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Cite this article as: Huang G, Liu J, Wang Y, Xiang Y. A study of the differential expression profiles of Keshan disease lncRNA/mRNA genes based on RNA-seq. Cardiovasc Diagn Ther 2021;11(2):411-421. doi: 10.21037/cdt-20-746